Characterization and functional analysis of the new microtubule associated protein RITA in the development of breast cancer

乳腺癌是全球关注的健康问题，也是全球女性死亡的主要原因。尽管取得了重大成就，乳腺癌转移在很大程度上仍然是无法治愈的。不受控制的细胞迁移和侵袭是转移过程的核心。微管在细胞迁移和侵袭中发挥时空调控作用。由于它们与多种调节蛋白和马达蛋白有关，它们与细胞迁移机制的不同组成部分相互作用，调节细胞极性、细胞内运输和局部黏附翻转。微管功能障碍导致恶性细胞不受控制地迁移，促进恶性肿瘤的发展。我们已经确定RBP-J相互作用和微管蛋白相关蛋白RITA在调节微管动力学和激活Aurora A方面至关重要，Aurora A是一种在各种肿瘤中经常被解除调控的激酶，这表明RITA参与了肿瘤的发生。事实上，RITA在肛门癌组织中高表达，其表达水平与患者的预后和治疗反应呈负相关。最近，我们报道了RITA与乳腺癌细胞的迁移/侵袭密切相关，它通过影响局部粘连的更新和细胞骨架的动力学来实现。然而，确切的分子机制仍不清楚。有趣的是，RITA与脂肪瘤首选伴侣(LPP)有关，LPP是乳腺癌细胞迁移和侵袭的重要参与者。通过这项建议，我们将研究RITA如何影响乳腺癌细胞的运动及其在乳腺癌发展中的意义。特别是，利用CRISPR/Cas9 RITA基因敲除/在细胞系、RITA基因敲除/敲除小鼠成纤维细胞、光漂白后荧光恢复、活细胞成像、转录本分析、原发乳腺癌组织以及其他分子/生化/细胞学方法，我们希望解决以下问题：1.RITA影响乳腺癌细胞迁移和侵袭的分子机制2.RITA和LPP在原发乳腺癌组织中的表达水平。3.乳腺癌组织中RITA的表达与肿瘤微环境及临床参数的相关性。鉴于RITA在调节微管动力学和参与肿瘤发生中的重要作用，本研究将提供去调控RITA影响乳腺癌发展、治疗反应和乳腺癌患者预后的分子机制。

Breast cancer, a global health concern, is the primary cause of death among women worldwide. Despite significant achievements, breast cancer metastasis remains largely incurable. Uncontrolled cell migration and invasion is central to the metastatic process. Microtubules exert spatiotemporal control in cell migration and invasion. Being associated with various regulatory and motor proteins, they interact with diverse components of the cell migration machinery and regulate cell polarity, intracellular trafficking and focal adhesion turnover. Dysfunctional microtubules result in uncontrolled migration of malignant cells and promote the development of malignancy. We have identified that RITA, the RBP-J interacting and tubulin associated protein, is crucial in regulating microtubule dynamics and the activation of Aurora A, a frequently deregulated kinase in various tumors, suggesting the involvement of RITA in oncogenesis. In fact, RITA is highly expressed in anal carcinoma tissues and its expression level is negatively correlated with prognosis and therapy response of patients. Recently, we report that RITA is tightly correlated with migration/invasion of breast cancer cells by affecting the turnover of focal adhesions and dynamics of the cytoskeleton. The precise molecular mechanisms remain however undefined. Interestingly, RITA is associated with the lipoma preferred partner (LPP), an important player in migration and invasion of breast cancer cells. With this proposal, we will investigate how RITA affects the motility of breast cancer cells and its significance in breast cancer development. In particular, using CRISPR/Cas9 RITA knockout/in cell lines, RITA knockdown/knockout mouse fibroblasts, fluorescence recovery after photo-bleaching, living cell imaging, transcriptomic analysis, primary breast cancer tissues as well as other molecular/biochemical/cellular methodology, we would like to address following issues: 1. The molecular mechanisms by which RITA affects migration and invasion of breast cancer cells 2. The expression levels of RITA and LPP in primary breast cancer tissues. 3. Correlation of the RITA expression with tumor microenvironment and clinical parameters of breast cancer patients. Given the crucial roles of RITA in regulating microtubule dynamics and its involvement in oncogenesis, this study will provide the molecular mechanisms by which deregulated RITA affects breast cancer development, therapy response and prognosis of breast cancer patients.